Vol. 61, Issue 5, 1211-1221, May 2002

Enhanced Detection of Receptor Constitutive Activity in the Presence of Regulators of G Protein Signaling: Applications to the Detection and Analysis of Inverse Agonists and Low-Efficacy Partial Agonists

Philip J. Welsby, Elaine Kellett, Graeme Wilkinson, and Graeme Milligan

Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (P.J.W., E.K., G.M.); and Molecular Pharmacology and Biochemistry, Department of Enabling Science and Technology, Astra-Zeneca, Alderley Park, Macclesfield, Cheshire, England, United Kingdom (G.W.)

Fusion proteins between the human 5-hydroxytryptamine (5-HT)_1A receptor and either wild type or certain pertussis toxin-resistant forms of G_o1 and G_i1 display constitutive GTPase activity that can be inhibited by the inverse agonist spiperone. Addition of recombinant regulator of G protein signaling (RGS) 1 or RGS16 to membranes expressing these fusion proteins resulted in elevation of this constitutive GTPase activity without significantly altering the binding affinity of antagonist/inverse agonist ligands. For a 5-HT_1A receptor-(Cys³⁵¹Ile)G_o1 fusion protein the increase in basal GTPase activity was greater than 4-fold. Enzyme kinetic analysis demonstrated that the effect of RGS1 was as a GTPase-activating protein for the fusion construct. In the presence of the RGS proteins, both agonists and inverse agonists produced much more robust regulation of high-affinity GTPase activity than in their absence. This allowed detection of the partial agonist nature of WAY100635, which has been described previously as a neutral antagonist at the 5-HT_1A receptor. Of a range of ligands studied, only haloperidol functioned as a neutral ligand in the presence of RGS1. These studies show that addition of a recombinant RGS protein provides a simple and novel means to elevate the fraction of basal membrane GTPase activity contributed by the constitutive activity of a receptor. By so doing, it also greatly enhances the ability to detect and analyze the effects of inverse agonists and to discriminate between neutral ligands and those with low levels of positive intrinsic efficacy.